1. Field of the Invention
The present invention relates, in general, to an immunogen for inducing antibodies that neutralize a wide spectrum of HIV primary isolates and to methods of inducing anti-HIV antibodies using this immunogen.
2. Description of the Background Art
Effective vaccination against HIV remains the only viable means to stop the spread of the AIDS pandemic. However, numerous attempts to elicit protective immunity to HIV have been unsuccessful.
The majority of antibodies generated against the HIV envelope glycoprotein gp120 are not neutralizing, either because their binding does not prevent fusion of HIV to its target cells or because the epitopes they recognize are inaccessible to the antibody. Therefore focusing the immune response to regions of gp120 that are known to bind neutralizing antibodies may improve the efficacy of vaccination. With few exceptions, even antibodies with neutralizing activity are only reactive against a limited number of HIV strains, a result of most antibody epitopes being subject to high sequence variability.
The V3 region of gp120, while generally variable, possesses conserved features that allow broad neutralization by certain antibodies such as the human monoclonal antibody (mAb) 447-52D (also referred to herein as 447-52 and 447). 447-52D recognizes the conserved tip of the V3 loop in a β-turn conformation.
However, most anti-V3 antibodies have narrow neutralization profiles. A specially designed V3-based immunogen that could induce high titers of antibodies with a binding mode and epitope specificity that is similar to that of one or more known broadly neutralizing antibodies (for example, 447-52D) is expected to be valuable as ah HIV vaccine.
Cholera Toxin subunit B (“CTB”) and a family of closely related bacterial proteins such as E. coli enterotoxins are homopentamers made of relatively small subunits (˜100 aa). The protein is highly immunogenic and has been used generally in fusion constructs to enhance immunogenicity of its fusion partner polypeptide or peptide. (McKenzie, S J et al., J Immunol 1984, 133:1818-24; Czerkinsky, C et al., Infec Immun 1989, 57:1072-7; Lipscombe, M et al., Mol Microbiol 1991, 5:1385-92). CTB has been described as a mucosal adjuvant for vaccines. Arêas A P et al., Biochem Biophys Res Commun. 2004, 321:192-6, genetically fused the ctxB gene to the psaA gene from Streptococcus pneumoniae, a surface protein, a vaccine antigen candidate. Purified CTB-PsaA expressed in E. coli, was used for intranasal immunization of mice and induced systemic and mucosal antibodies in serum, saliva, and in nasal and bronchial wash samples.
An important factor for the immunogenic property of CTB and related toxins is their binding to GM1 ganglioside. X-ray structures of CTB revealed that the oligosaccharide binding sites are formed by residues E51, Q56, H57, Q61, W88, N90, and K91 (Sixma T K et al., 1992, Nature 355; 561-4). The availability of this structural information allows protein design that avoids or minimizes disruption of the CTB GM1 binding site, thereby preserving the inherent immunogenicity of these polypeptides. Harakuni T et al., Infect Immun. 2005, 73:5654-65, disclosed that when used as a delivery means of a vaccine to mucosal immune systems, CTB cannot tolerate large-protein fusion which impairs pentamerization and lowers affinity for GM1-ganglioside. A new strategy to reduce steric hindrance between CTB-antigen fusion subunits promoted integration of unfused CTB “molecular buffers” into the pentamer unit, leading to more efficient self-assembly into biologically active pentamers. The chimeric protein took on a compact configuration, becoming small enough to be secreted. Affinity-purified proteins administered by a mucosal route induced specific immune responses in mice, a finding that was considered broadly applicable to bacterial enterotoxin-based vaccine design.
Its propensity to induce mucosal immunity is another advantage of CTB as an immunogenic “carrier” that is uncommon, yet is highly desirable for an HIV immunogen or vaccine because infection commonly occurs via a mucosal route. Furthermore, CTB is not toxic without the concomitant presence of the A subunit (that is part of the native cholera toxin. CTB has been approved as a component of an anti-cholera vaccine for use in humans.
In an attempt to generate an immunogen competent to generate 447-52D-like antibodies, Chakraborty et al., (Biochem. J. 399:483-91) inserted the known epitope of 447-52D at three different surface loop locations in the small, stable protein Escherichia coli thioredoxin (Trx). At one of the three locations (between residues 74 and 75), the insertion was tolerated (i.e., the resulting protein was stable and soluble) and bound 447-52D with an affinity similar to that of intact gp120. Upon immunization, with the V3 peptide-Trx scaffold, anti-V3 antibodies were induced that could compete for 447-52D binding to gp120. These anti-V3 antibodies were said to recognize the same epitope as 447-52D. The 447-52D-lik Abs were estimated to be present at concentrations of 50400 μg/ml of serum and were unable to effect neutralization of HIV-1 strains like JR-FL and BAL but could neutralize the sensitive MN strain. The authors suggested that because of the low accessibility of the V3 loop on primary HIV-1 isolates such as JRFL, it will be difficult to elicit a V3-specific, 447-52D-like antibody response to effectively neutralize such isolates.
It has also been observed that if the HIV-MN V3 epitope is placed in a scaffold, only strain-specific neutralization (of the MN strain) occurs. In contrast, the immunogenic compositions of the present invention surprisingly result in antibody responses that neutralize heterologous HIV-1 virus, as exemplified herein. Immunization with a combination of CTB+V3 as conceived by the present inventors was unexpectedly effective in inducing cross-clade antibody responses (see Examples).
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.